Serum Concentrations and Dietary Intake of Vitamin B12 in Children and Adolescents on Metformin: A Case–Control Study

The International Society of Pediatric and Adolescent Diabetes (ISPAD) recommends metformin (MET) use for metabolic disturbances and hyperglycemia, either in combination with insulin therapy or alone. A caveat of MET therapy has been suggested to be biochemical vitamin B12 deficiency, as seen mainly in studies conducted in adults. In the present case–control study, children and adolescents of different weight status tiers on MET therapy for a median of 17 months formed the cases group (n = 23) and were compared with their peers not taking MET (n = 46). Anthropometry, dietary intake, and blood assays were recorded for both groups. MET group members were older, heavier, and taller compared with the controls, although BMI z-scores did not differ. In parallel, blood phosphorus and alkaline phosphatase (ALP) concentrations were lower in the MET group, whereas MCV, Δ4-androstenedione, and DHEA-S were higher. No differences were observed in the HOMA-IR, SHBG, hemoglobin, HbA1c, vitamin B12, or serum 25(OH)D3 concentrations between groups. Among those on MET, 17.4% exhibited vitamin B12 deficiency, whereas none of the controls had low vitamin B12 concentrations. Participants on MET therapy consumed less energy concerning their requirements, less vitamin B12, more carbohydrates (as a percentage of the energy intake), and fewer fats (including saturated and trans fats) compared with their peers not on MET. None of the children received oral nutrient supplements with vitamin B12. The results suggest that, in children and adolescents on MET therapy, the dietary intake of vitamin B12 is suboptimal, with the median coverage reaching 54% of the age- and sex-specific recommended daily allowance. This low dietary intake, paired with MET, may act synergistically in reducing the circulating vitamin B12 concentrations. Thus, caution is required when prescribing MET in children and adolescents, and replacement is warranted.


Introduction
Metformin (1,1-dimethylbiguanide hydrochloride, MET) is considered the first-line oral blood glucose-lowering agent for the management of type 2 diabetes mellitus (T2DM) [1]. Although its use stems back to the Medieval European herbal medicine remedies of the plant Galega officinalis [2], the glucose-lowering properties of the plant were only discovered in 1918, attributed to its high guanidine content [3]. This propelled research on the use of guanidine derivatives, including MET, for the treatment of hyperglycemia. However, it was only in 1994 that the US Food and Drug Administration (FDA) first approved MET [1] due to the wide availability of insulin and the toxicity associated with guanidine use.
According to the American Diabetes Association (ADA), MET has been the drug of choice for the treatment of T2DM since the 1950s, carrying the "strongest evidence base" [4]. As for younger patients with T2DM, the International Society of Pediatric and Adolescent Diabetes (ISPAD) [5] recommends initial pharmacologic treatment with MET and insulin, either alone or in combination, depending on the degree of metabolic disturbances, as well as hyperglycemic and ketosis incidence. Furthermore, MET use can normalize the ovulatory abnormalities observed in girls with polycystic ovary syndrome (PCOS) [5]. More recently, research highlighted various MET-related extra-glycemic clinical benefits, including endothelial-protective [6], antineoplastic [7], and antiaging/anti-inflammatory effects [8]. Nonetheless, according to the ADA, long-term MET administration may be associated with biochemical vitamin B 12 deficiency [4], and this has also been mentioned in the ISPAD guidelines [5].
MET consists of a complex I mitochondrial inhibitor [nicotinamide adenine dinucleotide (NADH): ubiquinone oxidoreductase], transported inside the cell to influence cellular respiration [9]. Complex I oxidizes the NADH that is synthesized from the onecarbon metabolism, fatty acid β-oxidation, glycolysis, and the tricarboxylic acid (TCA) cycle for the production of adenosine triphosphate (ATP) through the transport chain of electrons [9][10][11]. Thus, biguanides induce a partial inhibition of the ubiquinone reduction [10], which increases the accumulation of NADH and the synthesis of reactive oxygen species (ROS), limiting the production of ATP, while increasing the adenosine monophosphate (AMP):ATP ratio [9]. In turn, the increasing concentrations of AMP:ATP stimulate the AMP-activated protein kinase (AMPK), inhibiting gluconeogenesis, while maintaining euglycemia [10].
MET has been shown to influence the status of several micronutrients, including vitamin B 12 and folate, both of which are important cofactors of the one-carbon metabolism [12,13]. In particular, MET use has been suggested to impair one-carbon metabolism similarly to anti-folate-class chemotherapy drugs administered for cancer [12,14]. Administration of MET reduces the intestinal absorption of vitamin B 12 , and the observed depression in vitamin B 12 concentrations disturbs the methylation cycle, increasing total homocysteine (tHcy) concentrations [9]. In parallel, MET's anti-folate activity also impairs the folate cycle [9]. Observational studies suggest that the administration of MET is associated with a small reduction in serum vitamin B 12 concentrations, although contradictory findings have also been reported in the literature [13,15,16]. Furthermore, most data stem from studies conducted on adults, with only a few using populations of younger patients [17,18]. Even more worrying is the fact that children and adolescents with overweight and obesity appear to be at greater risk for developing vitamin B 12 deficiency and exhibiting suboptimal vitamin B 12 status, irrespective of MET use [19]. In this manner, children with obesity on MET treatment may face a dual risk for exhibiting lower vitamin B 12 concentrations, as a result of the excessive body weight (BW) accumulation and the use of MET.
With this in mind, the present case-control study was designed to evaluate vitamin B 12 status (dietary intake and serum levels) in adolescents on MET treatment, compared with their peers who were not receiving MET. Table 1 details the results of the blood assays in each study group. No differences were noted in the median vitamin B 12 , 25-hydroxyvitamin D 3 [25(OH)D 3 ], fasting glucose, and insulin concentrations, glycosylated hemoglobin (HbA1c), or the homeostatic model assessment of insulin resistance (HOMA-IR) between groups. On the other hand, more participants on MET demonstrated biochemical vitamin B 12 deficiency compared with controls. Phosphorus and alkaline phosphatase (ALP) concentrations were lower in the MET group, whereas the ∆ 4 -androstenedione, mean corpuscular volume (MCV) and dehydroepiandrosterone sulfate (DHEA-S) concentrations were higher.  [20]; IQR-interquartile range; MCV-mean corpuscular volume; MET-metformin; P-phosphorus; SHBG-sex hormone-binding globulin. * Data are presented as medians with their respective first and third IQRs, or as counts (n) with their respective percentages (%); † vitamin B 12 concentrations <140 pg/mL; § Fisher's exact test. Table 2 presents the recorded daily dietary intake of participants. The consumption of energy and fats, including total monounsaturated fatty acids (MUFA), saturated fatty acids (SFA), and trans fats, was greater among participants in the MET group compared with controls. Concerning vitamin intake, differences were only noted in the intake of vitamins B 6 and B 12 , with a greater recorded intake among MET-receiving children, as well as concerning vitamin E, consumed in greater amounts than the controls. Iron, Magnesium, Zinc, and Sodium were also consumed in greater amounts by the controls.

Dietary Intake Results
The energy intake was suboptimal among controls, reaching 60% of their energy expenditure requirements (EER). Participants in either group failed to meet the recommendations for the intake of n-3 fatty acids, with those on MET therapy covering 39% (IQR 25.0-59.0) of their requirements and controls meeting 66.5% (IQR 49.0-90.5) of their needs. The dietary intake of vitamins B 9 , A, D, and E, Iron, Magnesium, Phosphorus, and Zinc was suboptimal among all participants, irrespective of group allocation. None of the participants in the MET-receiving arm reported taking oral nutrient supplements (ONS), whereas three participants (7%) belonging to the control group consumed multivitamin (MV) supplements.

Discussion
The present case-control study failed to show differences in the vitamin B 12 concentrations between children and adolescents on MET therapy compared with controls. However, more participants on MET demonstrated biochemical vitamin B 12 deficiency despite the greater reported dietary vitamin B 12 intake.
The recommendations for the parallel administration of vitamin B 12 in patients receiving MET were initiated from the results of several early case-control studies and clinical trials administering MET. One of these included the Diabetes Prevention Program (DPP) randomized controlled trial (RCT), where people with prediabetes used either MET or placebo [21]. A post hoc analysis of the data indicated a 13% increased risk of vitamin B 12 deficiency/year of MET administration after 13 years of follow-up [21]. In another placebo-controlled RCT, people with T2DM on insulin were randomized to MET as an add-on therapy or placebo for a total of 4 years [22]. The results revealed a 19% decrease in B 12 concentrations and a number needed to harm (NNH) of 13.8, over 4.3 years of followup [22]. Moreover, the reduction in B 12 concentrations was not transitory, but persisted and progressed over time [22]. With this in mind, the UK Medicines and Healthcare products Regulatory Agency (MHRA) published a new guidance identifying low B 12 concentrations as a distinct and common side-effect of MET therapy, especially among patients on highdoses or long-term treatment, estimated to affect up to one in 10 people [23]. In parallel, it recommends frequently checking B 12 concentrations in patients with possible symptoms of deficiency and closely monitoring those at risk of deficiency [23].
The present study revealed a greater prevalence of biochemical vitamin B 12 deficiency among children and adolescents on MET therapy compared to controls. Table 3 details the studies evaluating vitamin B 12 status in children/adolescents on MET treatment. The results appear controversial. Some studies reported a lack of change in the vitamin B 12 concentrations of minors on MET treatment [24][25][26], whereas others suggested that greater MET doses and prolonged treatment duration were associated with lower vitamin B 12 concentrations [17,18,[27][28][29], as suggested by the MHRA [23]. However, herein, the prevalence of vitamin B 12 deficiency greatly exceeded the rate proposed by the MHRA [23], with 18% of the sample treated with MET exhibiting total vitamin B 12 concentrations indicative of biochemical deficiency. BMI-body mass index; DSRA-dopamine and serotonin receptor antagonists; Holo-TC-II-holo-transcobalamin-II; IGT-impaired glucose tolerance; IR-insulin resistance; MET-metformin; MetS-metabolic syndrome; MMA-methylmalonic acid; MV-multivitamin; ONS-oral nutrient supplementation; PC-percentile; PCOSpolycystic ovary syndrome; RCT-randomized controlled trial; T1DM-type 1 diabetes mellitus; T2DM-type 2 diabetes mellitus; tHcy-total homocysteine.
The lack of consensus regarding the exact definition of vitamin B 12 deficiency is apparent in the scientific bubble [32,33]. The ongoing debate involves both the specific thresholds and the ideal biomarker (or combination of) to assess vitamin B 12 status accurately [32][33][34]. Suggested circulating vitamin B 12 biomarkers include total vitamin B 12 or holo-transcobalamin (HoloTC), whereas metabolic biomarkers of vitamin B 12 status involve the methylmalonic acid (MMA) or tHcy concentrations [35]. Several researchers [32,34,36] have underlined the limited diagnostic value of serum vitamin B 12 concentrations, due to its low sensitivity and specificity in identifying true tissue vitamin B 12 deficiency. In parallel, the assessment of total serum vitamin B 12 concentrations includes the circulating levels of the vitamin, 80% of which is bound to haptocorrin, limiting its cellular uptake bioavailability [32].
Research conducted on adults on MET therapy has also revealed important associations with specific methylene tetrahydrofolate reductase (MTHFR) polymorphisms. In particular, the C677T MTHFR defect has the potential to increase Hcy concentrations due to lower levels of methylcobalamin and methylfolate. In this manner, MET users harboring the rs180133 677C > T have been shown to attain suboptimal vitamin B 12 status, as well as greater Hcy and MMA levels [37,38]. For those carrying the C677T MTHFR variant, concomitant ONS with vitamin B 12 and methylfolate is recommended to correct for the low circulating levels.
MET has also been suggested to inhibit Ca-dependent absorption of the vitamin's B 12 intrinsic factor complex, at the site of the terminal ileum [39]. Since MET use reduces B 12 absorption through a Ca-dependent ileal membrane antagonism, Bauman [40] suggested that using Ca ONS can reverse the MET-induced serum vitamin B 12 and HoloTC depression. Nonetheless, in the present study, none of the participants in the NET-receiving arm were taking Ca ONS, while, on the other hand, Ca dietary intake was suboptimal for all participants.
Recent research revealed that children and adolescents receiving high levels of dietary methyl-donor nutrients (including vitamin B 12 ) have fewer chances of being metabolically unhealthy obese [41]. In parallel, vitamin B 12 levels are inversely related to the metabolic risk score of both children and their parents, through arterial blood pressure, high-density lipoprotein (HDLc) cholesterol, and triglyceride levels [42]. Other researchers have shown a negative relationship between obesity and vitamin B 12 concentrations [19] and an inverse association between IR and vitamin B 12 concentrations [43] in children and adolescents, irrespective of MET use. According to Infante [32], many conditions can increase the risk of vitamin B 12 deficiency, including inadequate dietary intake, impaired intrinsic factor secretion, malnutrition, vegetarianism, bacterial overgrowth syndromes, intestinal parasitic infestations, or disorders of the exocrine pancreas. All these factors should be evaluated prior to the initiation of MET therapy [32].
In the present study, participants in both groups reported a suboptimal dietary intake regarding several nutrients, with patients on MET therapy reporting the adoption of a more atherogenic diet. Furthermore, children and adolescents on MET treatment exhibited a greater consumption of dietary vitamin B 12 through food, without any intake of ONS. Thus, the lack of difference in serum vitamin B 12 concentrations between participants on MET therapy versus controls might well have resulted from increased dietary intake among the first. In parallel, it is also possible that the recorded greater vitamin B 12 consumption might have corrected possible lower levels, resulting from prolonged or high-dose MET therapy. Nonetheless, despite the greater intake, more children and adolescents on MET therapy demonstrated inadequate circulating vitamin B 12 levels compared to controls.
Concerning vitamin D concentrations, the present results confirmed previous studies on the fact that vitamin D status does not consist of a clinical concern among MET-treated patients [44,45].
An important limitation of the present study involves the relatively small sample size, as a larger population would probably have allowed reaching a significant difference in the concentration of vitamin B 12 between the two groups analyzed. The use of total serum vitamin B 12 concentrations as the only biomarker for the assessment of vitamin B 12 status is another limitation. Nonetheless, most of the studies available in the literature (Table 3) also relied on serum vitamin B 12 levels only [17,[24][25][26][27]29,31]. Furthermore, the diet record of participants is an important addition to the assessment of vitamin B 12 status, as most studies (Table 3) only relied on hematological parameters, ignoring the importance of dietary intake. Last, but not least, MTHFR polymorphisms were not assessed in the present population due to lack of consent by the parents, although they may well have impacted the observed associations.
Notably, suboptimal vitamin B 12 status is not the only adverse event associated with MET use. Gastrointestinal symptoms, bloating, flatulence, nausea, and diarrhea have also been reported and appear to be dose-dependent [46].

Study Population
During the first half of the year 2022, pediatric and adolescent outpatients were recruited randomly in a convenient manner from the Pediatric Endocrinology Unit of the Third Department of Pediatrics, situated at Hippokration General Hospital in Thessaloniki, Greece.
Those on MET treatment formed the case study arm, while those who were not on MET served as the controls of the study. Controls were also selected randomly from the children and adolescents visiting the Pediatric Endocrinology Unit, due to premature adrenarche, thelarche, precocious puberty, idiopathic short stature, microphallus, gynecomasty, overweight/obesity, hypothyroidism, or evaluation of thyroid dysfunction. Outpatients on MET (cases) were diagnosed with overweight/obesity, menstrual disorders/PCOS, and/or prediabetes/insulin resistance (IR). The ratio of cases versus controls was set at 1:2. Participant characteristics in each study group are presented in Table 4.  [47]; GH-growth hormone; IR-insulin resistance; MET-metformin; NS-not significant; PCOS-polycystic ovary syndrome. * Data are presented as counts (n) or means ± their respective standard deviations.

Ethical Permission
Permission for the study was granted by the Scientific Committee of the Hippokration General Hospital (4694/31-01-2023). In parallel, the parents/guardians of the participating children provided consent prior to their child's participation. The nature and purpose of the study were explained to all participants and their families by an experienced pediatric endocrinologist (K.T. and a dietitian E.G.P.) All data were handled with emphasis on anonymity and data protection, according to the Declaration of Helsinki and its latter amendments.

Anthropometric Measurements
The BW and stature of participants were measured to the nearest g and cm, respectively, using a Seca 700 mechanical scale (Seca, Hamburg, Germany) and a Harpenden wallmounted stadiometer (Holtain, Crymych, UK). All anthropometric measurements were performed in the morning by an experienced dietitian (E.G.P.).
Body mass index (BMI) was calculated for each participant as the ratio of BW (kg) to the square of height (m 2 ). BMI z-scores (BMIz) were calculated using the World Health Organization (WHO) Anthro software v.3.2 (WHO, Geneva, Switzerland) [48] for the assessment of growth and development of children and adolescents, based on the WHO child growth standards and growth curves [47,49].

Dietary Intake
For each child/adolescent, a detailed previous 24 h diet recall was collected with the facilitation of food photos with realistic sizes, and the intake was analyzed using the Cronometer software (Cronometer Software Inc., Vancouver, BC, Canada) [50].
In parallel, the intake of ONS was recorded for all participants, and each nutrient's daily intake was added to the respective recorded dietary intake.

Blood Samples and Assays
For biochemical and hormone profile analysis, fresh, whole-blood samples (20 mL) were collected from each participant in the morning hours, after overnight fasting. Plasma was isolated using ethylenediaminetetraacetic acid (EDTA). For serum isolation, whole blood was previously allowed to clot at room temperature for 20 min. Whole-blood samples were centrifuged at 3000 rpm for a total of 10 min at a temperature of 4 • C.
Immunoassay was performed via chemiluminescent detection for vitamin B 12 , total 25(OH)D 3 , insulin, sex hormone-binding globulin (SHBG), and dehydroepiandrosterone sulfate (DHEA-S). Insulin levels were assessed using a Human Insulin ELISA kit (ALPCO Diagnostics) with inter-and intra-assay precision below 15%.
The enzymatic method was used to assess blood glucose levels using an Abbot ALinity I analyzer. Ca, P, and ALP concentrations were estimated using Abbot ALinity C Analyzer (Abbott, Abbott Park, Chicago, IL, USA). Androstenedione was analyzed using an Immulite Siemens analyzer (Siemens Healthcare GmbH, Erlangen, Germany).

Insulin Resistance (IR)
Whole-body insulin sensitivity was evaluated with the homeostatic model assessment of insulin resistance (HOMA-IR) index in the fasting state [20], calculated as the fasting serum insulin concentrations (µU/mL) × plasma fasting glucose levels (mmol/L)/22.5.

Statistical Analyses
Normality in the distribution of variables was assessed using the Kolmogorov-Smirnov and Shapiro-Wilk tests. None of the variables appeared to follow a normal distribution. Qualitative variables were expressed as medians with their interquartile ranges (first and third IQR), and qualitative variables were expressed as counts (n) with their respective percentages (%).
Between-group comparisons were conducted using the Mann-Whitney U or the chisquared test. Fischer's exact test was applied to compare frequencies between the two groups.

Conclusions
The rises in the prevalence of childhood and adolescent overweight and obesity [52][53][54] and its associated comorbidities have increased the number of youngsters receiving MET therapy [55]. Overall, MET consists of a low-cost option with modest clinical benefits for BW loss and minimal side-effects [56,57]. Thus, when lifestyle treatment has been pursued as a first-line therapy and deemed suboptimal in achieving adequate weight loss, a reasonable continuum would be using MET, as an adjunctive therapy [56]. Nonetheless, MET treatment appears to affect vitamin B 12 status in children and adolescents, irrespective of dietary intake. According to the recent clinical practice guidelines published by the American Academy of Pediatrics [58], when prescribing such medications, healthcare professionals must adequately inform patients and their parents about the risks and benefits of therapies and must have updated knowledge of the patient selection criteria, medication efficacy, possible adverse events, and the recommendations regarding follow-up monitoring. For this, frequent assessment of vitamin B 12 concentrations and recording of symptoms and signs associated with vitamin B 12 deficiency are warranted when prescribing MET to children and adolescents. In parallel, currently, there is no evidence supporting weight loss or diabetes medication use as a monotherapy [58]. In this manner, lifestyle treatment must be prescribed as an adjunct to MET therapy. Informed Consent Statement: Written informed consent has been obtained from all parents/guardians of the participating children and adolescents. Data Availability Statement: All data are available to the corresponding author upon request.